Detergent containing alkali protease

ABSTRACT

Alkaline protease if produced by cultivating an alkaline protease-producing microorganism from the genus Fusarium or Giberella in a proper growth supporting medium and the alkaline protease is recovered from the medium. The alkaline protease degrades protein under conditions of high pH and thus is useful in the formulation of detergent and other cleanser compositions.

United States Patent Isono et al.

[is] 3,655,570 1451 Apr. 11,1972

DETERGENT CONTAINING ALKALI PROTEASE lnventorsi Masao I sono,Nishinomiya; Katsumi Tomoda, Toyonaka, Osaka; Kouichi Miyata, Takatsuki,Osaka; ,Kazutaka Maejima, Nishinomiya; Keisuke Tsubaki, Suita, Osaka,all of Japan Assignee: Takeda Chemical Industries, Ltd., Osaka,

- Japan Filed: Feb. 3, 1969 Appl. No.: 795,951

Foreign Application Priority Data Feb. 8, 1968 Japan ..43/7863 Mar. 15,1968 Japan ..43/l6970 U.S. Cl ..252/132, 252/89, 252/135 Int. Cl. ..Clld7/42, Cl ld 9/40 [58] Field ofSearch 195/62, 66, 63, 68; 252/135,252/DIG. 12

References Cited UNITED STATES PATENTS 3,451,935 6/1969 Roald et al..252/1 35 Primary Examiner-Lionel M. Shapiro Attorney-Wenderoth, Lind &Ponack [5 7] ABSTRACT 5 Claims, 6 Drawing Figures PATENTEDAPRH 1912' I3,655,570

'SHEET 2 OF 5 (70) HDNVLLIWSNVHL MASAO ISONO KATSUMI ToMbDA,

KOUICHI MIYATA,

KAZUTAKA MAEJIMA &

KEISUKE TSUBAKI INVENTORS Attorneys PATENTEDAPR n 1912 3,555,570

' SHEET u 0F 5 Fig. 5

MASAO I SONO KA'ISUMI TOMODA,

KOUI CHI MIYATA KAZUTAKA MAEJIMA AND KEISUK E TSUBAKI Inventors Bywwm:AM

Attorneys PATENTEDAPR 11 191? 3. 655,570

SHEET 5 BF 5 Fig. 6

TEMPERATURE ('C) All/\LLOV 'IVOOISHU MASAO ISONO,

KATSUMI TOMODA,

KOUICHI MIYATA,

KAZU'IAKA MAEJIMA' AND KE'ISUKE TSUBAKI,

Inventors By, MMM,%LL

Attorneys DETERGENT CONTAINING PROTEASE The present invention relates toa novel alkali protease and to a process for producing the protease aswell as to detergents and other cleansers containing the enzyme.

in the course of study for obtaining alkali proteases, it'has been foundthat certain microorganisms belonging to the genus Fusarium or the'genusGibberella produce an alkali protease which exhibits a potentproteolyticactivity around It has been found that the said proteaseactively degrades various kinds of protein under conditions of highpHeven in the presence of'surfactantsand/or chelating agents,'suggestingits applicabilityin the field of the detergent industry. 'l-leretofore,although certain enzymes'havebeen applied to' the laundry process,satisfactory results have not=always been obtained. One of the possiblecauses-therefor is th'at most cleansing solutions have a'pH over pl-l8.Another cause is that the enzymic activity 'is sometimes inhibited'by'surfactants and/or chelating agents contained in the-detergents.

"In thisconnection, further extensive studies-have been carried out'anda process established forjproducing a potent al 'kali protease ona'cornmercial'scale and manufactured detergents and other clean'serscontaining' the enzymes'lhe enzyme is referred toherein as alkaliprotease.

"The principal object of the'present'invention is therefore-to providean alkali proteasewhich exhibitspotent activityin the p'l-l r'arlge'from8:0 to 1210, particularly, from 10.0 to 1 1 .5.

Another object is toprovidedetergents and other cleansers containing'thealkaliprotease.

ln order 'to' realize the objects of the present invention,'-a "strainof alkali protease-producing microorganisms belonging to the genusFusariumorthe genus Gibberellais cultivated in a culture medium. Some;typical-=microoganisms' producing the alkaliprotease are as'follows:

Fusariumoxysporum (ll- 05942) ('ATCC659) Fusari'umoxysporum fibatatas(IFO 4468) 'Fusarium oxysporum f. gladioli- "(IFO 5894) Fusariumoxysporumfllini (IFO 5880) Fusariumoxysporumf; neveum IFO 4471)Fusarium-bxysporumf.*liriilvasinfectum :(IFO4472) Fusarium solani (lFO5232) Fusarium-spl s:1'9:5 (IFO8884) ATCC 20l92) {Gibberlldfujikuroi n=o 5268) I I I Gibberella saubin'etii 1 (lF'6608) (ATCC 20193 lFO numbersin the parentheses are the accession numbers at the institute forFermentation, Osaka, Japan; ATCCnumb'e'rs-areaccession numbers atAmerican Type Culture Collec- "'tion Rockville', Maryland.

* Amongthese microogranisms,"Fusarium sp'.' S'-19-5-is one of the "mostuseful strains for the production of jthe alkali protease. This strainwas isolated from a soil-sample-"by the inhyphae; wrinkled on reverse; ascarlet-purple pigment r 'in themedium. 3'. Czapeckssolution:

On each of the above media, conidiophores develop from aerial hyphae,unbranched, 10 to along, 1.0 to 1.5 uwide, hyaline. Conidia are producedapically on conidiophore and grouped in a slimy cluster, ovate orkidnay-shaped, one to two-celled, rarely three-celled, 5 to 8 I by l to2 p), hyaline. -No sexual state develops. Chlamydospore usually absent,sometimes present. 3. Physiological characteristics: 1. Condition forgrowth:

Hydrogen ion'concentration: Preferably grows in alkaline media,optimalat between pH 8.0 to 9.0 Temperature; Optimal at about 24 C. Allow togrow'between -l5 and 28 C.,-although better growth is observed around at15 C. rather than at 28 C. Oxygen requirement: Aerobic. 2. Gelatin:liquefaction:

Slight. 3; Utilization of ethyl-alcohol:

Negative. 4. Degradation of pectin:

Very slight. 5 Degradation of tannin:

Negative. 6 Degradation of fats and oils:

Positive. 7 Utilization of carbohydrate:

Utilizes maltose, r'galactose, melibiose, sucrose, trehalose, fructose,.mannose, raffinose, dextrin, starch,1glucoseand lactose. Slightlyutilizes inulin, xyloseparabinose and cellulose. According to Dictionaryof theFungi (G.C. Ainsworth, 5th edition), the said microbialcharacteristics indicate that this particular strain'belongs to thegenus Fusarium, the family 'luberculariaceae, the order Moniliales, thesubclass Deutermycetes, the class Fungi Imperfecti.

Based on the Snyder&Hansen classification,

i-Eusarium is divided into Section 1fifl 2 follows:

: A rmicroconidia develop, usually one-celled.

A no microconidia develop or rarely produced, spindle,

comma'or kidney-shaped, one to'several-celled. The organism, Fusariumsp.-S-l9--5,' produces microconidia --dominantly and can be included inth Elegans Group of SectionA. The Group Elegans'isitypified by F usariumoxysporum and includes 36 species which are all plant pathogensproducing macroconidia in abundance.

"The organism, Fusarium sp. S-l9-5, however, produces only microconidiain mostcases andthe formation of macroconidiais very rare. Thischaracteristic indicates the or- #:ganism cannot be assigned'to any ofthe hitherto known species of'this group. 7 v

In order to cultivate analkali protease-producing microorganism, per seconventional culture media and conditions are used.

-itis more advantageous to adopt shake culture or aerobic cul- -turemethods.

a The medium may be usedin either liquid or solid form. The culturemaybe effected under stationary conditions, however,

"ple, it may contain, as carbon sources, such materials as glucose,sucrose, dextrin, starch, cellulose glycerol, sorbitol and Good growth,-white; flocculant pellicle formed; wrinkled on reverse; substantially nopigment. 4YCz apecks agar:

* Good growth, white and-flocculant; wrinkled onreverse;

substantially no pigment. 5. Gelatin:

Good growth, white and flocculant; dark blackish purple pigmentonreverse; weak liquefaction. 6." Potato dextrose:

" Goodgrowth, white and flocculant;-the surface uneven,

raised; wrinkled on reverse,- white tolight purple; pur- 'ple pigmentliberated in the medium. 2; Microscopic characteristics:

the-like, and, as nitrogen sources, such materials as peptone,

meatextract, yeast extract, dried yeast, soybean meal,

soybean cake, rice'bran, wheat bran, potato extract, casein,

gluten, casein hydrolysate, corn steep liquor, urea, ammonium salts,nitrates and other organic or inorganic nitrogenous compounds. Asinorganic salts, various phosphates, sulfates and hydrochlorides, forexample, may be incorporated. Under certain circumstances, for thepurpose of promoting growth of "the microogranism, various vitamins,amino acids, nucleic acids and their related compounds, etc. may beadded. Depending upon the culture methods and conditions to be employed,the addition of a natural or synthetic defoarning agent, e.g., soybeanoil or silicone oil, may be effective to increase accumulation of theenzyme.

ln cultivating the microorganisms, it is preferable to prepare a smallscale preculture which is, in turn, inoculated into a main culturemedium.

Culture conditions such as incubation temperature and time, pH of themedium, aeration rate, etc., should vary with the microorganism andmedium compositions to be used.

The requisite is that the conditions should be selected and controlledso that the accumulation of the alkali protease is maximal. In manyinstances, the preferred conditions include the incubation temperatureof from 20 to 30Co., an incubation time from 2 to 7 days, a medium pH ofnear 7, and an aeration rate of 0.5 to 1.5 liters per minute per literof the medium.

Thus, the microogranism accumulates a large amount of the alkaliprotease in the culture. When a liquid medium is employed, the objectenzyme occurs mostly in the liquid phase of the culture. Therefore, itis preferable to follow the steps of removing mycelia by filtration orcentrifugation and, then, of recovering and isolating the enzyme fromthe filtrate or the supernatant fluid as the case may be.

When a solid medium is employed, it is usually preferable to subject theculture to extraction with water or an aqueous solution of an inorganicsalt and, then, to recover the enzyme from the resulting extract.

In order to recover and isolate the enzyme from the culture filtrate,supernatant or extract, any of per se conventional isolation andpurification means may be employed. These include salting-out of enzymewith an inorganic salt such as sodium sulfate, ammonium sulfate orsodium chloride as well as fractional precipitation of enzyme by addinga suitable hydrophilic organic solvent such as methanol, ethanol oracetone. In addition, an enzyme solution may be concentrated underreduced pressure and/or demineralized by dialysis. It is also possibleto employ means such as adsorption and desorption on calcium phosphategel, alumina, bentonite, ion exchange resin, etc., chromatography usinga cellulose derivative, e.g. diethylaminoethyl cellulose,gel-filtration, precipitation, electrodialysis, electrophoresis andremoval of impurities as heavy metal complex.

The alkali protease produced and prepared in the foregoing manner isactive over a broad pH range between 8.0 and 12.0, particularly in thepH region of 10.0 to l 1.5.

The optimal temperature for enzyme activity lies somewhere between about20 and about 60 C., particularly between about 40 and about 50 C. Thistemperature range for the activity is in quite good accord with actuallaundry conditions. Moreover, the activity of the alkali protease is notinhibited by surfactants and/or chelating agents which are principalingredients in detergent products or cleansers.

In this invention, the alkali protease may be employed not only as ahighly purified preparation but as a crude enzyme product and thus theproduct is obtained in a desired purity in accordance with the purpose;either pure or crude preparation of the enzyme may be used as aningredient of detergent products or cleansers.

As surfactants contained in the detergent products, there may bementioned for purpose of exemplification various compounds includinganionic surfactants of the fatty acid salt type, sulfate type orsulfonate type, such as natural fatty acid soap (NS), alkylsulfate(DAE), olefine sulfate, n-a-olefine sulfonate (AOS), tetrapropylbenzenesulfonate (ABS) and n-alkylbenzene sulfonate (LAS); and non-ionicsurfactants of the ether type or ester type, such as polyoxyethylenealkyl ethers, polyoxyethylene alkylphenol ethers, polyhydric alcoholalkyl esters, polyoxyethylene alkyl esters, sugar esters and the like.

The detergent products or cleansers may contain builders such astri-polyphosphate, sulfates, carbonates, borates, as well ascarboxymethyl cellulose, fluorescent dyes, scents, bleaching agents,e.g. perborates, chelating agents, e.g., N(CH COONa);;, skin-protectiveagents, e.g. dimethyllaurylaminoxide, disinfectants, e.g. tertiarilyamine, and

the like. he

The alkali protease is mixed with other components of the product whichmay be a powdery, granular or liquid form and,

when the resulting mixture is a liquid, it may be dried to a powdery orgranular product by conventional means such as p y y s- Since there isno international system of unit for this kind of enzyme, it is difficultto prescribe the portion of the enzyme in detergent products orcleansers in general.

For purposes of the present invention, activity of the alkali proteaseis assayed by the following modified Kunitz method (J. Gen. Physiol. 30,291, 1947). 1.0 ml. of a 2 percent auqeous solution of casein(I-Iammarsteins) and 0.5 ml. of an NaOI-I buffer, pH 11.0, are mixedwith 0.5 of an enzyme solution. 2.0 ml. of the resulting mixture isallowed to incubate at 37 C. for 20 minutes, and then the reaction isstopped by the addition of 3.0 ml. of a 5 percent aqueous solution oftrichloracetic acid. The mixture is allowed to stand at 37 C. for afurther 30 minutes, whereby the undigested casein is thoroughlyprecipitated. The precipitated casein is filtered off and the resultingfiltrate is subjected to optical density determination at 275 mu, fromwhich the amount of the digested casein is calculated as the amount oftyrosine. One unit of protease activity (PU) is defined as the amount ofthe enzyme which dissolved an amount of casein equivalent to 1.0 pg. oftyrosine per minute under the assay conditions,. The specific activityof an enzyme sample is expressed as PU/mg.

The portion of the alkali protease to be incorporated in detergentproducts will vary with the type of product. In the case of a detergentto be used for washing cloth, the preferred amount will generally rangefrom 5 to 10,000 in terms of units per gram of the detergent and forpractical purposes from 10 to 5,000 units per gram.

An alkali protease-containing detergent prepared in the foregoing mannershows exceedingly powerful cleansing action against proteinous stainsatributable to sweat, blood, broth and milk, which are hard to wash awaywith a conventional detergent.

Further, the detergent is employed in prewashing as well as inmainwashing.

In prewashing, soiled materials are usually soaked in a detergentsolution at a room temperature for several hours, while the mainwashingis preferably carried out at an elevated temperature between 40 and 60C. for 1 hour or so.

Presently preferred embodiments of the invention are shown in thefollowing examples, but they are not intended to be construed as alimitation on the present invention.

Throughout the specification, the abbreviations ml, mg., p.g,p., mu andC." mean milliliter(s), milligram(s), microgram(s), micron(s),millimicron(s) and degree centigrade, respectively. Percentages arecalculated on the weight per volume basis. In the following examples,parts by weight bear the same relation to parts by volume as do gram(s)to milliliter(s).

EXAMPLE I 500 parts by volume of a liquid medium, composed of 5 percentdefatted soybean meal, 5 percent glucose, 2 percent sodium dihydrogenphosphate, and adjusted to pH 7 is dispensed in a fermenter (itscapacity being 2,000 parts by volume), sterilized, inoculated withFusarium sp. S-l9-5 (IFO 8884) (ATCC 20192) and incubated at 28 C. for 5days under aeration and agitation to prepare a seed culture.

The seed culture is inoculated to a fermenter (its capacity being 50,000parts by volume) containing 30,000 parts by volume of the same liquidmedium as above, and the fermenter is incubated at 25 C. for 144 hourswith the aeration rate of 45,000 pars by volume per minute underagitation of 500 rev./min. During the incubation, foaming is suppressedby the addition of a suitable amount of soy bean oil from ime to imChanges of the pH value and the protease activity course of thecultivation are shown as follows:

Time of culture (hours) 18 30 42 54 66 78 90 122 144 pH of culture 7.306. 40 6.30 6.12 6.35 6.72 7. a0 7. 09 7. 50 1,50 Enzyme activity(PU/ml.) 67 141 1,430 2,250 2. 270 2, 520 2, 520 2. 760 2,540

EXAMPLE 2 Soluble in water and aqueous mineral salt solutions hav- Theculture obtained after 144 hours, as in Example 1, is $25235 s figfifiiogginzerlnsoluble 1n cooled to about C. and, then, passed through afilter press 9 pH activity (See FIG with the filter aid, Hyflo Super-Ce](Johns-Manville Products optimal PH at about 11 Corp. U.S.A.), wherebythe mycelia is removed. To the result- Temperature activity s FIG ing20,000 parts by volume of the filtrate is added 0.6-satu- Optimaltemperature about 5 rated ammonium sulfate, and the salted-outprecipitate is col- 1 1 pH Stability (See FIG. 5 lected by filtrationwith the filter aid. The resulting ammonium N i tivation in the pH rangefrom 5 to 8; 10 percent sulfate precipitate containing the filter aid isdissolved in i ti i n t H 11.5 (1 hour-incubation at 37C.). about 6,000parts by volume of cold water and insoluble 12. Temperature stability(See FIG. 6): materials are removed by filtration. 0.6-Saturatedammonium No inactivation below 55 C; percent inactivation at sulfate isthen added to the filtrate, as to precipitate the en- 65 and 80 percentat 70 C. (10 minute-incubation at zyme again, which is, in turn,collected by centrifugation, dispH 5). solved in 1,000 parts by volumeof cold water, dialyzed against 20 EXAMPLE 4 cold water by means offish-skin diaphragm for 4 days and lyophilized to give a crude enzymepowder. By the above procedure, 30 parts by weight of the crude enzymepowder with brownish color is obtained. The specific activity of thissample is bout 980 PU/mg.

EXAMPLE 3 1n 3,000 parts by volume of cold water is dissolved 30 partsby weight of the crude enzyme powder prepared in Example 2, andinsoluble materials in the enzyme solution are filtered off with thefilter aid to obtain a clear solution. 0.6-Saturated ammonium sulfate isadded to the clear filtrate to give precipitate of the enzyme which is,then, re-dissolved in 1,500 parts by volume of cold water, decolorizedwith charcoal, dialyzed against cold water at 45 C. for 4 days andliphilized to a powder. The above procedure yields 4.5 parts by weightof a partially purified enzyme powder with a specific activity of 1,580PU/mg.

4.5 parts by weight of the enzyme powder, dissolved in 450 parts byvolume of 0.01 M Tris-HCl buffer, pH 9.0, are dialyzed against 0.001 MTris-HCl buffer, pH 9.0, for 3 days put on a column of diethylaminoethylcellulose previously equilibrated with the Tris-HCl buffer and elutedwith the same buffer. About 675 parts by volume of the enzyme richfraction are collected, dialyzed against cold water for 3 days andlyophilized to a powder. The above procedure yields 0.35 part ofpurified enzyme in a powdery state with a specific activity of 5,400PU/mg.

0.35 Part by weight of the purified enzyme powder, dissolved in 335parts by volume of 0.01 M Tris-HCl buffer., pH 8.0, is chromatographedthrough a column of Sephadex G-100 previously washed with the TrisHClbuffer. 62 parts by volume of the active fraction is collected, dialyzedagainst cold water for 3 days and lyophilized, whereupon 0.15 part byweight of a highly purified enzyme powder is obtained. The specificactivity of this sample is 6,500 PU/mg.

The enzymological characteristics of this example are as follows:

1. Color and shape:

White powder. 2. A typical elementary analysis C, 42.72; H, 6.59; N,15.03. 3. Sedimentation constant (S about 3.19 X 10. 4. Molecular weight(by Archibalds method): 3.65 X 10 (15%). 5. Ultraviolet absorptionspectrum (see FIG. 1):

Maximal absorption at 275-280 mu; 280nm,tcm 6. Infrared absorptionspectrum (See FIG. 2):

Significant absorption bands in microns at 3.38, 6.05,

6.55, 6.88, 7.15, 8.12, 9.30. 7. lsoelectric point (by paperelectrophoresis):

About pH 11. 8. Solubility:

Influence of several surfactants on the alkali protease is examined asfollows:

According to the formula given in Table l, five different detergentcompositions are prepared where sodium salt of n-C natural fatty acid(NS), sodium n-C -alkylsulfate (DAS), sodium n-a-C olefmesulfonate(AOS), sodium tetrapropylbenzenesulfonate (ABS) and sodium n-C-alkylbenzenesulfonate are used as the surfactants, and these result- 5Mg. each of the respective detergents is dissolved in 2 ml. of an enzymesolution prepared by dissolving 2 mg. of the crude enzyme powderobtained in Example 2 into ml. of 0.05 M Tris-HC1 buffer, pH 1 1.0, andthe enzyme activity of the resulting solution is assayed according tothe specified assay method. The results are shown in Table 2 whichindicates that none of these detergents inhibits the enzyme activity.

Enzyme-containing detergent compositions are prepared by blending 0.05g. of the crude enzyme powder obtained in Example 2 with 100 g. each ofthe above different detergents.

TABLE 3 Prepared by the method of Japan Oil and Fat ChemicalAssociation, using a 1:1 mixture of air cleaner dust and casein in placeof carbon black.

Soiled Cloth (5 cm X cm) The cleaning activity is evaluated by a panelmonitation. Five pieces of the soiled cloth are assigned to the test ofeach detergent and, after the sequence of washing, rinsing and dryingunder the specified laundry test, the resulting washed cloths are scoredby five judges according to the following standards of score.

Standards of score:

+2 Cleanliness of the cloth washed with an enzyme-containing detergentis definitely superior to that with the corresponding enzyme-freedetergent.

+1 Cleanliness of the cloth washed with an enzyme-containing detergentis slightly superior to that with the corresponding enzyme-freedetergent.

0 No significant difference is observed in cleanliness between thecloths.

The results of scoring are shown in Table 4, where the relativecleansing effects of the enzyme-containing detergents versus thecorresponding enzyme-free detergents are presented, as to the respectivetypes of detergent.

In the same manner as in Example 1, several microogranisms belonging tothe genus Fusarium and the genus Gibberel- 1a are cultivated for 6 days.

The cultures are then centrifuged to give supernatant fluids which areused as enzyme solutions. To 2,000 parts by volume each of the enzymesolutions are added 5 parts by weight of the LAS-detergent described inExample 4, and protease activity of the resulting solution is determinedby the specified assay method. The results are set forth in Table 5indicating that the LAS-detergent does not inhibit any activity of theenalkali zymes produced by those protease-producing microogranisms.

TABLE 5 Enzyme Activity (PU/ml) Mircoogranism with without detergentdetergent Furan'um vxysporum (ll-O 5942) (ATCC 659) 200.8 199.2 Furariumuxyrporumf. lini (ll-O 5880) 550.5 553.8 Furarium oxyrporum f. niveum(IFO 4471) 315.8 307.2 Fusarium .rvlani (IFO 5232) 230.2 235.0

Gibberella fujikuroi (IFO 5268) 73.2 75.6 Gibberella .ruubinelti (II-O6608) (ATCC 20193) 530.2 523.4

EXAMPLE 7 A liquid detergent for kitchen use: In 55 parts by volume ofhot water at 60 65 C. are dissolved 18 parts by weight of sodiumtetrapropylbenzenesulfonate, 12 parts by weight of sodium n-Calkylphenolether-sulfate, 5 parts by weight of lauryldiethanolamide and10 parts by weight of sodium xylenesulfonate. After allowing to cool,the solution is supplemented with 0.5 part by weight of the crude enzymepowder prepared in Example 2 and a small quantity of a scent, to give aliquid detergent composition for kitchen use.

EXAMPLE 8 Hair shampoo: In 64 parts by weight of hot water at 60-65 C.are dissolved 5 parts by weight of acetylated lanolin, 6 parts by weightof Alkylolarnine (American Alcolac Corp.) and 25 parts by weight ofDuponol XL E. I. duPont de Nemours Company). After allowing to cool, thesolution is supplemented with 0.2 part by weight of the crude enzymepowder prepared in Example 2 and a small quantity of a scent, to give ahair shampoo.

What is claimed is:

l. A detergent or cleanser composition comprising at least onesurfactant and alkali protease selected from the group consisting ofFusarium sp. S-19-5 (ATCC 20192), Fusarium oxysporum f. lini (IFO 5880)and Gibberella saubinetti (ATTC 20193), characterized by the followingproperties:

1. sedimentation constant (s of about 3.19 X 10",

2. molecular weight of about 2.65 X 10 (by Archibalds method,

3. elementary analysis about 46.72 percent by weight carbon, about 6.59percent by weight hydrogen and 15.3 percent by weight nitrogen,

4. maximum absorption, in its ultraviolet absorption spectrum at thewavelength of 275 to 280 u, as indicated in FIG. 1,

5. infrared absorption spectrum as shown in FIG. 2, wherein thesignificant absorption bands in microns are as follows: 3.0 (strong),3.38 (medium), 6.05 (strong), 6.55 (strong), 6.88 (weak), 7.15 (medium),8.12 (medium), 9.30 (weak),

6. isoelectric point about pH 1 l,

7. optimal activity at a pH from 8 to 12 as indicated in FIG. 3, and ata temperature from about 40 to about 50 C. as indicated in FIG. 4,

8. stable in the pH range from 5 to 8 in 1 hour-incubation at 9. 20 andpercent loss of activity upon being heated at 65 and 70 C.,respectively, in 10 minute-incubation at pH 5.0.

2. A composition as in claim 1, wherein the amount of the said alkaliprotease is from about 5 to about 10,000 PU per gram of the finalproduct by the modified Kunitz method.

3. A composition as in claim 1, wherein the said surfactant is selectedfrom an anionic surfactant and a nonionic surfactant.

4. A composition as in claim 3, wherein the said anionic surfactant isfrom the group of a fatty acid salt, alkyl sulfate, olefine sulfate,alkyl sulfonate, olefine sulfonate, and alkylaryl sulfonate.

5. A composition as in claim 3, wherein the said nonionic surfactant isfrom the group of polyoxyethylene-alkyl ether, polyoxyethylene alkylphenol ether, polyoxy-ethylene alkyl ester, polyhydric alcohol alkylester and sugar ester.

2. molecular weight of about 2.65 X 104 (by Archibald''s method,
 2. A composition as in claim 1, wherein the amount of the said alkali protease is from about 5 to about 10,000 PU per gram of the final product by the modified Kunitz'' method.
 3. A composition as in claim 1, wherein the said surfactant is selected from an anionic surfactant and a nonionic surfactant.
 3. elementary analysis about 46.72 percent by weight carbon, about 6.59 percent by weight hydrogen and 15.3 percent by weight nitrogen,
 4. A composition as in claim 3, wherein the said anionic surfactant is from the group of a fatty acid salt, alkyl sulfate, olefine sulfate, alkyl sulfonate, olefine sulfonate, and alkylaryl sulfonate.
 4. maximum absorption, in its ultraviolet absorption spectrum at the wavelength of 275 to 280 Mu , as indicated in FIG. 1,
 5. infrared absorption spectrum as shown in FIG. 2, wherein the significant absorption bands in microns are as follows: 3.0 (strong), 3.38 (medium), 6.05 (strong), 6.55 (strong), 6.88 (weak), 7.15 (medium), 8.12 (medium), 9.30 (weak),
 5. A composition as in claim 3, wherein the said nonionic surfactant is from the group of polyoxyethylene-alkyl ether, polyoxyethylene alkyl phenol ether, polyoxy-ethylene alkyl ester, polyhydric alcohol alkyl ester and sugar ester.
 6. isoelectric point about pH 11,
 7. optimal activity at a pH from 8 to 12 as indicated in FIG. 3, and at a temperature from about 40* to about 50* C. as indicated in FIG. 4,
 8. stable in the pH range from 5 to 8 in 1 hour-incubation at 37* C.,
 9. 20 and 80 percent loss of activity upon being heated at 65* and 70* C., respectively, in 10 minute-incubation at pH 5.0. 